Reproduction depends on the orchestration of LH and FSH biosynthesis and release by neuroendocrine, endocrine, autocrine and paracrine stimuli. Mouse studies show that both GnRH and activin/TGF? signaling are needed for Fsh?, which is required in the mouse for female reproduction. Thus reproduction depends on a GnRH/activin ?AND? logic gate having an unknown molecular basis. Relevant to this problem, our recent studies support a new working model for gene regulation in the gonadotrope. Using single cell (sc) RNA quantification we find that gene induction to different GnRH concentrations is all-or-none, with genes showing a characteristic increased probability of turning on in single cells at higher concentrations of GnRH . Thus what we consider transcriptional regulation can be modeled as probabilistically activated binary on-off switches at the sc level. Differences in regulatory mechanisms detected at sc resolution and evidence for gonadotrope subtypes indicate the importance of sc transcriptome and epigenetic assays in extending these studies to gonadotrope regulation in vivo. Based on a probabilistic gene control model, recent findings and review of the literature, we hypothesize that the GnRH/activin Fsh? gate results from relative effects of activin and GnRH signaling on the immediate probability of Fsh? gene activation and on the promoter accessibility state, most likely by activin signaling predominantly increasing Fsh? gene accessibility and GnRH increasing the immediate probability of activation. Accordingly, we propose in Aim 1 to study the separate and combination effects of activin and GnRH and to test the probabilistic all-or-none (quantal) model in L?T2 cells using bulk ATAC-seq chromatin accessibility assays and sc integrated fluidics circuit mRNA assays. In Aim 2, we will characterize the relationship of chromatin landscape and gene expression in male mice and in female mice and identify gonadotrope subtypes and their regulatory processes through the estrous cycle using sc RNA-seq and sc ATAC-seq assays. In Aim 3 we will use sc RNA-seq and sc ATAC-seq to study mechanisms responsible for the Fsh? gate using GnRH and activin-signaling deficient mouse models. These studies will be interpreted using state-of-the-art bionformatic frameworks and, when necessary, by modifying or developing new methods. While these studies are designed to test specific hypotheses, the use of genome-wide discovery platforms will provide data for mining and new hypothesis generation, such as understanding diverse autocrine/paracrine regulatory mechanisms and will generate a resource for the field. The proposed research program should provide fundamental insight into the molecular identity, heterogeneity and regulation of gonadotropes and an improved understanding of the mechanisms for the gonadotrope gene control mechanisms that underlie reproduction.